Biosensor arrays and methods

ABSTRACT

A surface detector array device suitable for use with a biosensor is disclosed. The device is formed of a substrate having a surface defining a plurality of distinct bilayer-compatible surface regions separated by one or more bilayer barrier regions. The bilayer-compatible surface regions carry on them, separated by a film of aqueous, supported fluid bilayers. The bilayers may contain selected receptors or biomolecules. A bulk aqueous phase covers the bilayers on the substrate surface.

This application is a continuation-in-part of U.S. patent applicationSer. No. 08/978,756 filed Nov. 26, 1997, now U.S. Pat. No. 6,228,326,which claims benefit of U.S. Provisional application Serial No.60/032,325 filed Nov. 29, 1996, both of which are entirely incorporatedherein by reference.

This application further claims the benefit of priority to U.S.provisional applications No. 60/205,604, filed May 18, 2000, nowabandoned, and 60/158,485, filed Oct. 8, 1999, now abandoned, both ofwhich are entirely incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates in general to supported fluid bilayers andmethods of confining them to selected areas. More specifically, theinvention relates to microfabricated arrays of independently-addressablesupported fluid bilayer membranes and their uses.

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BACKGROUND OF THE INVENTION

Over the last several years, a number of high-throughput screeningmethods have been developed to facilitate the screening of thousands, ifnot millions, of compounds for a desired activity or activities. Suchmethods are typically based on detecting the binding of a potentiallyeffective compound to a receptor. While these binding assays areeffective at constraining the universe of compounds which may have thedesired activity, they are typically not well-suited for evaluating thisactivity with any degree of detail.

The biological activity of potentially active compounds is typicallyevaluated using less efficient but more informative “secondary screens”or assays which typically require a substantial input of time by atrained technician or scientist. For evaluation of candidate compoundsaffecting integral membrane proteins such as receptors and ion channels,the amount of time required per compound may be several hours or days ifthe assay includes effects on electrophysiological activity.Accordingly, there is a need for a more efficient “secondary screen” ofcompounds affecting the activity of such integral membrane proteins, toidentify those few compounds that justify further detailed analysis.

SUMMARY OF THE INVENTION

In one aspect, the present invention includes a surface detector arraydevice. The device includes a substrate having a surface defining aplurality of distinct bilayer-compatible surface regions separated byone or more bilayer barrier regions, a bulk aqueous phase covering thesubstrate surface, a lipid bilayer expanse carried on each of thebilayer-compatible surface regions, and an aqueous film interposedbetween each bilayer-compatible surface region and corresponding lipidbilayer expanse. In a general preferred embodiment, thebilayer-compatible surface regions and the bilayer barrier surfaceregions are formed of different materials.

The bilayer-compatible surface region may be formed from any of avariety of materials having such bilayer-compatible surface properties,including SiO₂, MgF₂, CaF₂, and mica, as well as a polymer film, such asa polyacrylamide or dextran film. SiO₂ is a particularly effectivematerial for the formation of a bilayer-compatible surface region.

The bilayer barrier surface region may be formed from any of a varietyof materials having such bilayer barrier surface properties, includinggold, positive photoresist and aluminum oxide.

In a general embodiment, the lipid bilayer expanse contains at least onelipid selected from the group consisting of phosphatidylcholine,phosphatidylethanolamine, phosphatidylserine, phosphatidic acid,phosphatidylinositol, phosphatidylglycerol, and sphingomyelin.

In one embodiment, the device contains between about 10 and about 100distinct bilayer-compatible surface regions. In another embodiment, thedevice contains at least about 2500 distinct bilayer-compatible surfaceregions. In yet another embodiment, the device contains at least about25,000 distinct bilayer-compatible surface regions. In still anotherembodiment, the device contains at least about 2.5 million distinctbilayer-compatible surface regions.

The bilayer-compatible surface regions are separated from one another,in one general embodiment, by bilayer barrier regions that are betweenabout 1 μm and about 10 μm in width.

The lipid bilayer expanses on different bilayer-compatible surfaceregions may have different compositions, and may further include aselected biomolecule, with different expanses having a differentbiomolecule, such as transmembrane receptor or ion channel. Thebiomolecule may be covalently or non-covalently attached to a lipidmolecule. Examples of non-covalent interactions include electrostaticand specific molecular interactions, such as biotin/streptavidininteractions. Examples of biomolecules include proteins, such as ligandsand receptors, as well as polynucleotides and other organic compounds.

In another aspect, the invention includes a method of forming a surfacedetector device having a plurality of independently-addressable lipidbilayer regions. The method includes the steps of (i) treating a planarsubstrate to form a substrate surface defining a plurality of distinctbilayer-compatible surface regions separated by one or more bilayerbarrier regions, and (ii) applying a suspension of lipid bilayervesicles to the plurality of distinct bilayer-compatible surface regionsunder conditions favorable to the formation of supported bilayers on thebilayer-compatible surface regions. The applying then results in theformation of supported bilayer membranes on the bilayer-compatiblesurface regions.

In yet another aspect, the invention includes a method for detecting aselected ligand in a mixture of ligands. The method includes the stepsof (i) contacting the mixture with a biosensor surface detector arraydevice such as described above, and (ii) detecting binding of theselected ligand to receptors which specifically bind it.

In still another aspect, the invention includes a surface detectionarray device for use in a biosensor. Such a device includes (i) asubstrate having a surface defining a plurality of distinctbilayer-compatible surface regions separated by one or more bilayerbarrier regions, (ii) a bulk aqueous phase covering the substratesurface, (iii) a lipid bilayer expanse carried on each of thebilayer-compatible surface regions, and (iv) an aqueous film interposedbetween each bilayer-compatible surface region and corresponding lipidbilayer expanse. Each bilayer expanse contains a species of receptor orbiomolecule, and different bilayer expanses contain different species ofreceptors or biomolecules.

Another aspect of the present invention provides for a surface detectorarray device, comprising a substrate having a surface defining aplurality of distinct bilayer-compatible surface regions separated byone or more bilayer barrier regions, a bulk aqueous phase covering saidsubstrate surface, a lipid bilayer expanse carried on each of saidbilayer-compatible surface regions, and an aqueous film interposedbetween each bilayer-compatible surface region and corresponding lipidbilayer expanse, wherein said bilayer-compatible surface regions andsaid bilayer barrier surface regions are formed of different materials,and wherein each bilayer-expanse carried on each bilayer-compatibleregion is compositionally different than adjacent bilayer-expanses.Other embodiments of the invention further include a plurality of groupsof said bilayer-compatible regions, wherein said groups each define anarea where said bilayer-expanses are compositionally similar, and wherethe bilayer-expanses within different groups are compositionallydifferent.

The invention further provides a method for forming an array ofbiosensor regions, where each region has a different, known lipidbilayer composition comprising the steps of:

providing a biosensor array having a plurality of lipid bilayercompatible regions, each compatible region being surrounded by one ormore bilayer barrier regions,

providing a gradient forming devices loaded with two or more differentlipid bilayer compositions, the gradient forming device in fluidcommunication with a spot forming device for forming spots on a surface,

providing a multi-axis translation table for holding and translating abiosensor array workpiece,

placing a biosensor array workpiece that has a plurality of bilayercompatible regions surrounded by one or more barrier regions, and

forming spots of mixed lipid bilayer compositions resulting from thegradient forming device forming a gradient and translating the table inat least one axis while dispensing such composition mixture as it isformed thereby dispensing to different, consecutive locations differentratios of each lipid bilayer compositions.

The invention further provides a method for making gradient biosensorarray comprising the steps of:

mixing together first and second different lipid bilayer formingcompositions contained from first and second sources by flowing in asubstantially laminar flow, two different compositions from twodifferent sources into one mixing chamber that substantially retains thelaminar flow character of the two different compositions while flowingthrough the mixing chamber, where the facing edges of each differentcomposition mix to form a gradient having a first edge and a second edgeand further comprising composition combinations of different ratiosbeginning from the first edge of the gradient that faces the firstcomposition, and ending at the second edge of the gradient that facesthe other, second composition, and where the mixing chamber is adaptedto dispense the gradient in a substantially laminar flow across thesurface of the array, and

where the compositions contained in the gradient are captured andretained upon initial contact by bilayer-compatible regions of thearray.

These and other objects and features of the invention will become morefully apparent when the following detailed description is read inconjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a portion of a surface detector array device (SDAD) of theinvention.

FIGS. 2A and 2B are schematics illustrating the effect of photobleachingfluorescent reporter lipids in the lipid bilayers of five distinctregions of a surface detector array device of the invention.

FIG. 3 shows the fluorescence intensity from two regions of a surfacedetector array device, each containing a field-induced concentrationgradient of charged fluorescent reporter lipids.

FIG. 4 shows the structural portion of a device of the inventionsuitable for use in a biosensor.

FIG. 5 shows the structural portion of a device of the inventionsuitable for use in separating membrane-associated molecules by size.

FIG. 6 depicts a top down view of the forming of a gradient biosensorarray.

FIGS. 7a-d depict in color the formation of a two composition gradientbiosensor array.

DETAILED DESCRIPTION OF THE INVENTION I. Definitions

The terms below have the following meanings unless indicated otherwise.

The term “aqueous” refers to a water-based liquid medium that is notdeleterious to lipids.

A “receptor” is a macromolecule capable of specifically interacting witha ligand molecule. In cells, receptors are typically associated withlipid bilayer membranes, such as the extracellular, golgi or nuclearmembranes. Receptors for incorporation into expanses of lipids in vitro(e.g., supported bilayers) may either be purified from cells,recombinantly expressed, or, in the case of small receptors, chemicallysynthesized.

A “ligand” is a molecule capable of specifically binding to a receptor.Binding of the ligand to the receptor is typically characterized by ahigh binding affinity, i.e., K_(m)>10⁵, and can be detected either as achange in the receptor's function (e.g., the opening of an ion channelassociated with or part of the receptor) or as a change in the immediateenvironment of the receptor (e.g., detection of binding by surfaceplasmon resonance). Ligands for incorporation into expanses of lipids invitro (e.g., supported bilayers) may either be purified from cells,recombinantly expressed, or, in the case of small ligands, chemicallysynthesized.

Binding is “specific” if it results from a molecular interaction betweena binding site on a receptor and a ligand, rather than from“non-specific” sticking of one protein to another. In cases where theligand binds the receptor in a reversible manner, specificity of bindingcan be confirmed by competing off labeled ligand with an excess ofunlabeled ligand according to known methods. Non-specific interactionscan be minimized by including an excess of a protein (e.g., BSA) thatdoes not have binding sites for either the ligand or receptor.

II. Surface Detector Array Device

FIG. 1 is a perspective view of a portion of a surface detector arraydevice (SDAD) 20 in accordance with the invention.

The device is fabricated from a substrate 22, such as an oxidizedsilicon or fused silica wafer. The dimensions of the substrate aretypically between about 0.5 cm to about 5 cm per side and about 0.1 mmto about 1 cm in thickness.

The substrate surface contains a plurality of distinctbilayer-compatible surface regions 24 separated by one or more bilayerbarrier regions 26. The bilayer barrier region(s) 26 are preferablyformed of a material 28 different from the material 22 forming thebilayer-compatible surface regions 24.

A lipid bilayer expanse 30 is carried on each of the bilayer-compatiblesurface regions 24. Interposed between each bilayer-compatible surfaceregion 24 and corresponding lipid bilayer expanse 30 is an aqueous film32 that is between about 5 Å and 15 Å (typically about 10 Å) inthickness. Covering the substrate surface and lipid expanses is a bulkaqueous phase 34.

The bilayer barrier regions may be depressed, flush, or elevated (asshown at 26 in FIG. 1), with respect to the bilayer-compatible surface24. In embodiments having elevated barriers, the height of the barriermay range from tens of Angstroms to several micrometers or more. Thewidth of the barriers is typically between about 100 nm and about 250μm. Preferably, the width is between about 1 and 100 μm.

According to results of experiments performed in support of theinvention, the lipid barrier regions do not function simply bymechanical or physical separation of adjacent lipid bilayer regions.Rather, the experiments indicate that the characteristics which allow asurface to act as a bilayer barrier region are chemical/electrostaticproperties intrinsic to the material making up the surface. Examples ofsuch chemical/electrostatic properties include hydrophobicity,dielectric permeability, conductivity, and surface charge density.

Similarly, the degree of “bilayer-compatibility” of a selected surfaceis a function of its intrinsic material properties rather than itsshape. The interactions between membranes and surfaces involveelectrostatic and hydration forces as well as attractive contributionsfrom long-range van der Waals forces. In a suitable bilayer-compatiblesurface, an energetic minimum traps the bilayer membrane between about 5Å and 15 Å (typically about 10 Å) away from the supporting surface,separated from the supporting surface by an aqueous film ofcorresponding thickness. Bilayer-compatible surfaces are typicallyhydrophilic.

Functionally, the suitability of a material for use as a bilayer barriersurface region or a bilayer-compatible surface region may be evaluatedby the material's performance in a simple “fluorescence recovery afterphotobleaching” (FRAP) test as follows:

A small sample of the material (e.g., a portion having a ˜1 cm² flatsurface) is cleaned or treated as described herein (e.g., using exposureto argon plasma or, for materials which can tolerate it, an acids wash).The surface is then rinsed and a selected amount (e.g., 50 μl) of asuspension of lipid vesicles containing a fluorescent marker (preparedas described in the Materials and Methods) is applied to the surface.The suspension is allowed to remain in contact with the surface forseveral minutes (e.g., 5 min). The surface is then immersed in anaqueous medium to rinse off or substantially dilute the suspension(e.g., by adding ˜100 ml of distilled water or PBS), and the surface istransferred to the stage of a standard fluorescence microscope. Aportion of the surface is then exposed to a bright light (e.g, from a100W mercury arc lamp) sufficient to bleach the fluorescent moieties ofthe reporter exposed to the light (e.g., about 1 min., depending on thefluorophore), and the surface is monitored under the microscope for ˜10minutes (depending on size of the bleached spot) to assess recovery offluorescence.

When the above test is carried out using a material capable of forming abilayer-compatible surface, vesicles in the suspension will have fusedwith the surface forming a supported bilayer containing the fluorescentreporter, and the localized exposure to photobleaching light will havebleached the area of the bilayer corresponding to the region of thesurface on which the photobleaching light was focused. During themonitoring period, fluorescence in the bleached area of the bilayer willrecover due to the fluidity of the supported bilayer.

In contrast, when the above test is carried out using a material thatforms a bilayer-barrier surface, vesicles in the suspension will nothave fused with the surface to form a fluid bilayer. Under suchconditions, the vesicles will either be rinsed off during the rinse stepor will remain attached and immobilized on the surface. If the vesiclesrinse off, little or no fluorescence will be observed. If the vesiclesstick to the surface but do not form a fluid bilayer, fluorescence willnot be recovered in the bleached area following photobleaching. Ineither case, the material is an effective bilayer barrier material. Itwill be appreciated, however, that the use of materials to which thevesicles do not stick is preferable to the use of those which, followingthe above FRAP test, contain immobilized lipid or membrane material.

The above test can be carried in parallel with a number of differentmaterials that the practitioner of the invention may have at hand. Inthis way, in a matter of a few hours, the practitioner can readilydetermine whether a particular material will be effective to form asurface that is either bilayer-compatible or serves as a bilayerbarrier.

It will be appreciated that essentially all materials suitable for usein the microfabrication of a device according to the invention will,when cleaned, present either a bilayer-compatible surface region or abilayer-barrier surface region. Accordingly, application of the simpleFRAP test described above will typically yield a material useful in thepractice of the invention with each material tested.

Exemplary materials having properties making them suitable for lipidbilayer barriers include certain polymers (e.g., photoresist) andvarious metals (e.g., gold) and minerals (e.g., aluminum oxide). Anadvantage of photoresist is that it is relatively easy to pattern with aphotomask and is non-conductive. Aluminum oxide has the advantage ofbeing both nonconductive and reusable, withstanding most cleaningprocedures.

Exemplary materials having properties making them suitable forbilayer-compatible surfaces include various glasses, silicon oxides,including oxidized silicon (SiO₂), MgF₂, CaF₂, mica, and various polymerfilms, such as thin polyacrylamide or dextran films (see, e.g., Elender,et al., 1996; Khüner, et al., 1994), both incorporated herein byreference). Both types of polymer films form a suitablebilayer-compatible surface that is hydrated to provide a film of aqueousbetween the polymer film and the supported bilayer membrane.

To generate a substrate surface that is “bilayer-compatible”, thesurface is typically cleaned and/or treated to remove surface impurities(dirt, oils, etc.). Suitable treatments are discussed below with respectto the making or construction of a device of the invention.

The supported bilayer itself is a self-assembling, two-dimensional fluidsystem, typically consisting of two opposed leaflets of vesicle-forminglipid molecules. However, it can be constructed as described below fromany suitable membrane-forming amphiphile, including proteins andnonlipids.

Most vesicle-forming lipids are long-chain carboxylic acids, such asglycerides, having the hydroxyl groups of the glycerol esterified with(i) fatty acid chain(s), and (ii) a charged or polar moiety, such as aphosphate-ester group. The vesicle-forming lipids are preferably oneshaving two hydrocarbon chains, typically acyl chains, and a polar headgroup. Long-chain carboxylic acids with a phosphate group, orphospholipids, are particularly well-suited for use with the presentinvention.

There are a variety of synthetic vesicle-forming lipids andnaturally-occurring vesicle-forming lipids, including the phospholipids,such as phosphatidylcholine (PC), phosphatidylethanolamine (PE),phosphatidylserine (PS), phosphatidic acid, phosphatidylinositol (PI),phosphatidylglycerol (PG), and sphingomyelin, where the two hydrocarbonchains are typically between about 14-22 carbon atoms in length, andhave varying degrees of unsaturation. The above-described lipids andphospholipids whose acyl chains have varying degrees of saturation canbe obtained commercially or prepared according to published methods.Other suitable lipids include glycolipids and sterols such ascholesterol.

Preferred diacyl-chain lipids for use in the present invention includediacyl glycerol, phosphatidyl ethanolamine (PE) and phosphatidylglycerol(PG). These lipids are preferred for use as the vesicle-forming lipid,the major liposome component, and for use in the derivatized lipiddescribed below. All of these phospholipids and others are availablefrom specialized suppliers of phospholipids (e.g., Avanti Polar Lipids,Inc., Alabaster, Ala.) as well as from general chemical suppliers, suchas Sigma Chemical Co. (St. Louis, Mo.).

The aqueous film and bulk aqueous phase may be any suitable aqueoussolution, such as a buffered saline solution (e.g., PBS). The bulksolution can be readily changed (taking care, of course, to keep thesupported bilayer submerged at all times) by, e.g., flow-through rinsingwith a solution having a different composition.

As described above, FIG. 1 shows a support grid microfabricated from awafer of a material which forms the bilayer-compatible surfaces of thedevice. A device may also be microfabricated, however, from a wafer of amaterial which forms the bilayer-barrier surface regions of the device.One embodiment of such a device is shown in FIG. 4. Here, the structuralportion 50 of a device of the invention is produced by microfabricatinga wafer of a bilayer barrier material 52 (e.g., aluminum oxide) tocontain regions, such as region 54, consisting of a bilayer-compatiblematerial, where each region corresponds to one of the plurality ofdistinct bilayer-compatible surface regions, such as region 56. In oneembodiment, the regions 54 are electrically-conductive and are connectedto leads 58 which can be used to record changes in the membranepotential at the surface. An example of an electrically-conductivebilayer-compatible material is a metal, such as gold, coated with a thinfilm of silicon oxide or polymer material to make the surfacebilayer-compatible. The thin film of silicon oxide, while not anelectrical conductor, can effectively pass capacitative current.

Alternatively or in addition, electrodes having a bilayer-compatiblesurface may be generated from standard doped (e.g., boron-doped) siliconwafers. A layer of silicon oxide may be formed on such wafer substratesto provide a bilayer-compatible surface, under which resides asemi-conductor (doped silicon) electrode. The semi-conductor electrodecan, of course, be interfaced with any of a variety of other elements,e.g., semi-conductor elements in the substrate itself or in a separatechip, as desired, to facilitate or enhance the processing of informationfrom the patch of bilayer membrane corresponding to that electrode.

A number of different devices have been produced in accordance with theinvention. They include the following (i) a device containing a 1 cm²array of 2500 identical 200 μm square corrals or regions, (ii) a devicecontaining a 1 cm² array of 10,000 identical 100 μm square regions,(iii) a device containing a 1 cm² array of about 37,000 identical 50 μmsquare regions separated by 2 μm barriers of photoresist, and (iv) adevice containing a 1 cm² array of about 2.8 million 5 μm square corralsor regions separated by 1 μm-wide barriers of photoresist.

Exemplary embodiments of the invention include devices where the bilayerlipid expanses contain different biomolecules, such as receptor proteinmolecules, ligand protein molecules, or other protein molecules. Suchdevices are particularly useful in biosensors, described more fully inthe applications section of the specification, and are made as describedbelow by fusing proteoliposomes to the bilayer-compatible surface.

It is recognized that proteoliposome vesicles can be fused to a glasssurface to create a planar supported membrane (Brian and McConnell,1984). This technique has been successfully applied in a number ofsituations. In one example, the H-2K^(k) protein was reconstituted intoegg phosphatidylcholine-cholesterol vesicles by detergent dialysis, andthe vesicles were used to create a planar membrane on glass (Brian andMcConnell, 1984). The H-2K^(k)-containing membrane was capable ofeliciting a specific cytotoxic response when brought into contact with acell.

Chan, et al. (1991) demonstrated that a glycosylphosphatidylinositol(GPI)-anchored membrane receptor is laterally mobile in planar membranesformed from proteoliposome fusion, and that this mobility enhances celladhesion to the membrane. Other applications employ a combination ofvesicle fusion, Langmuir-Blodgett methodology and derivatized surfacesto prepare supported membranes (Sui, et al., 1988; Plant, et al., 1995).

In addition to incorporation of receptors or ion channels into thebilayer membrane, the bilayer may be derivatized with any of a number ofgroups or compounds to create a surface having the desired properties.For example, the liposomes may contain a ligand bound to the surface ofthe lipid by attachment to surface lipid components. Generally, such aligand is coupled to the polar head group of a vesicle-forming lipid.Exemplary methods of achieving such coupling are described below.

III. Construction of a Surface Detector Device withIndependently-Addressable Lipid Bilaver Regions

Surface detector device of the invention may be conveniently producedusing a combination of microfabrication and lipid vesicle technologies,e.g., as described in Example 1.

A. Microfabrication of Patterned Support Grid

Patterning of the substrate to produce a substrate surface having aplurality of distinct bilayer-compatible surface regions separated byone or more bilayer barrier regions may be done in a number of differentways appreciated by those knowledgeable in the microfabrication artshaving the benefit of the present specification. For instance,micromachining methods well known in the art include film depositionprocesses, such as sputtering, spin coating and chemical vapordeposition, laser fabrication or photolithographic techniques, oretching methods, which may be performed by either wet chemical or plasmaprocesses. These and other micromachining methods are summarized, forexample, in Petersen (1982), incorporated herein by reference. Generalsilicon processing techniques known in the art are described, forexample, in Wolf and Tauber (1986) incorporated herein by reference.

A device is typically produced by first selecting a substrate materialand producing a patterned support grid (the structural portion of asurface detector array device of the invention). The support gridcarries on the patterned side the substrate surface according to theinvention. The substrate is typically of a material selected to have theproperties of one of either a bilayer-compatible or bilayer barriermaterial with strips of a material having the properties of the other ofa bilayer-compatible or bilayer barrier material. In one generalembodiment, a bilayer-compatible substrate material is patterned withstrips of a bilayer-barrier material. In another general embodiment, thesubstrate material is a bilayer barrier material and its surface ispatterned with regions of a bilayer-compatible material. It will beappreciated, however, that the substrate material can be patterned withboth regions of bilayer-compatible material and regions of bilayerbarrier material, such that the original substrate material is notrepresented at the patterned substrate surface. The materials which doform the substrate surface are selected such that after surface cleaningand/or treating, one yields a bilayer-compatible surface region and theother yields a bilayer barrier surface region.

Photoresist has at least two potential uses with respect to the presentinvention. As discussed above, positive photoresist is an effectivebilayer-barrier material. Of course, photoresist can also be used in thetraditional sense of patterning a substrate for subsequent lithographyto generate microfabricated devices of the invention. Suitablenegative-or positive-resist materials are well known. Commonnegative-resist materials include two-component bisarylazide/ rubberresists, and positive-resist materials include two-componentdiazoquinone/phenolic resin materials. An example of electron beamresist, which may also be suitable, includes polymethylmethacrylate(PMMA) see, e.g., Thompson, et al. (1983).

As mentioned above, silicon is a preferred substrate material because ofthe well-developed technology permitting its precise and efficientfabrication, but other materials may be used, including polymers such aspolytetrafluoroethylenes. The substrate wafer (e.g., silicon wafer) istypically cleaned using a standard RCA clean (Kern and Puotinen, 1970;Wolf and Tauber, 1986). The wafer is then oxidized at a temperature ofbetween about 800 and 1000° C. in steam using known methods (Wolf andTauber, 1986) until a layer of oxide (preferably about 0.5 □m inthickness) is formed. The oxide layer is then coated with a photoresistlayer preferably about 1 □m in thickness. As described herein, thismethod can be used to produce the structural portion of an exemplarysurface detector array device of the invention, which now only needs tobe cleaned as described below before it is exposed to a vesiclesuspension to generate the bilayer expanses.

Alternatively, the photoresist-patterned substrate can be subjected tostandard photolithography to produce a surface detector array devicewith a material other than photoresist forming the bilayer-barrierregions. In this case, the coated laminate is irradiated through aphotomask imprinted with a pattern corresponding in size and layout tothe desired pattern. Methods for forming photomasks having desiredphotomask patterns are well known. For example, quartz plates can bepatterned with chrome with electron beam machine and an electron beamresist, such as PBS, using standard methods. Alternatively, a mask canbe obtained commercially from any of a number of suppliers, e.g.,Align-Rite (Burbank, Calif.). Exposure is carried out on a standardcontact mask aligner machine, such as a Karl Suss contact lithographymachine. Conventional positive or negative photoresists may be used withclear-field or dark-field photomasks. The pattern may be transferred tothe substrate by subsequent etching or liftoff processes.

Electrodes may be fabricated into the device using any of a number ofdifferent techniques are available for applying thin metal coatings to asubstrate in a desired pattern. These are reviewed in, for example,Krutenat, 1986; and in Wolf and Tauber, 1986, both incorporated hereinby reference. Convenient and common techniques used in fabrication ofmicroelectrodes include vacuum deposition, evaporation, sputtering, andplating. Various conductive materials, including doped silicon andmetals such as platinum, gold, or silver may be used for the electrodes.

Deposition techniques allowing precise control of the area of depositionare preferred for application of electrodes to the selected regions ofthe device. Such techniques are described, for example, in Krutenat,above, and in Wolf and Tauber. They include physical vapor depositionusing an electron beam, where atoms are delivered on line-of-sight tothe substrate from a virtual point source. In laser coating, a laser isfocused onto the target point on the substrate, and a carrier gasprojects powdered coating material into the beam, so that the moltenparticles are accelerated toward the substrate.

Another technique allowing precise targeting uses an electron beam toinduce selective decomposition of a previously deposited substance, suchas a conventional electron beam resist (e.g., PMMA), a thin layer ofanother material (e.g., a metal salt), a monolayer, or the like (see,e.g., Tiberio, et al., 1993). This technique has been used to producesub-micron circuit paths (e.g., Ballantyne, et al., 1973). It will beappreciated that the dimensions of the different regions can be madeextremely small, since electron beam lithography along with near fieldscanning microscopy may be used to generate and image membrane patternson the nanometer scale. Further, certain non-traditionalmicrofabrication materials having bilayer-barrier properties can bepatterned using standard technologies. For example, aluminum oxide canbe patterned on SiO₂ substrate wafers by evaporation and liftoff (Wolfand Tauber, 1986, see p. 535). Such patterning, as well as the generalmicrofabrication described above, can be conveniently done bycontracting the work out to a company offering microfabricationservices, such as MCNC (Research Triangle Park, N.C.), IC Sensors(Milpitas, Calif.) and Silica-Source Technology (Tempe, Ariz.).

B. Cleaning of Patterned Support Grid

After the patterned support grid is made, it is cleaned and/or treatedto strip or etch off any impurities or contaminants present on thesubstrate surface which might otherwise inhibit the formation of a lipidbilayer adjacent the surface. The cleaning procedure is selected suchthat it does not substantially damage the functionality of the bilayerbarrier regions. For example, embodiments where the barrier regions aremade of photoresist should not be cleaned using the traditional pirhanasolution acid wash (3:1 H₂SO₄:H₂O₂), since the acid can strip off thebilayer barrier regions. An exemplary cleaning/treating process thatdoes not damage photoresist employs exposure of the patterned grid toargon or oxygen plasma for several minutes. Although the plasma doesetch the photoresist somewhat, it strips off contaminants from thesurface layer of the substrate (e.g., SiO₂ substrate) beforesubstantially damaging the photoresist layer.

A number of suitable etching and/or cleaning procedures are known in theart. Four such procedures are summarized below. They include thosedescribed above and may be employed separately or in combination. In thefirst method, the structural portion of the device (support grid) isbaked at 500° C. for several hours. This method is not compatible withgold or photoresist. In the second method, the support grid is washed inpirhana solution acid wash (3:1 H₂SO₄:H₂O₂). This method is notcompatible with photoresist and many metals, although it can besuccessfully used with gold and platinum. In the third method, thesupport grid is boiled in detergent (e.g., 7×detergent from ICNBiomedicals, Inc. (Aurora, Ohio), diluted 1:4). This method is notcompatible with photoresist and is not very effective used alone. In thefourth method, the support grid is etched in a gas plasma (e.g., argonor oxygen). This method works most effectively when combined with thethird method, but can be used alone; it is the only procedure describedherein that is suitable for use with photoresist.

C. Making Supported Bilayer Expanses

Following such a wash/etching/treatment step, the grid is placed in achamber and a suspension of vesicles or liposomes formed of a selectedlipid and (optionally) containing selected proteins or otherbiomolecules is contacted with each bilayer-compatible surface region.Vesicles in the suspension generally fuse with the bilayer-compatiblesurface region within minute or less to form a supported bilayermembrane (Xia, et al., 1996; Groves, et al., 1996). A humidified chamberis preferably used in applications where the volume of the drops oflipid suspension is small enough (e.g., ˜<5 μl) to allow substantialevaporation before the bilayers form and the grid is flooded with bulkaqueous.

Liposomes may be prepared by a variety of techniques, such as thosedetailed in Szoka, Jr., et al. (1980). The lipid components used informing liposomes useful in making the present invention preferablycontain at least 70 percent vesicle-forming lipids. In one generalembodiment, the bilayers are formed as described in Example 1.

As discussed above, the supported bilayers may contain receptors ofother biomolecules, such as peptides, nucleic acids, factors, etc.,attached to or incorporated into the supported bilayer membrane. Methodsfor producing such “modified” bilayers using “derivatized” liposomes, orliposomes containing an additional moiety such as a protein, are wellknown (see, e.g., Zalipsky, 1995; Allen, et al., 1995, as well as U.S.Pat. Nos. 6,605,630, 4,731,324, 4,429,008, 4,622,294 and 4,483,929). Afew examples are discussed below.

One procedure suitable for preparation of such derivatatized liposomesinvolves diffusion of polymer-lipid conjugates into preformed liposomes.In this method, liposomes are prepared from vesicle-forming lipids asdescribed, and the preformed liposomes are added to a solutioncontaining a concentrated dispersion of micelles of polymer-lipidconjugates. The mixture is then incubated under conditions effective toachieve insertion of the micellar lipids into the preformed liposomes.

In another method, the biomolecule is coupled to the lipid, by acoupling reaction described below, to form an biomolecule-lipidconjugate. This conjugate is added to a solution of lipids for formationof liposomes, as will be described. In another method, a vesicle-forminglipid activated for covalent attachment of a biomolecule is incorporatedinto liposomes. The formed liposomes are exposed to the biomolecule toachieve attachment of the biomolecule to the activated lipids. In yetanother method, particularly suitable for making liposomes containingintegral membrane receptors or proteins, the liposomes are simply formedin the presence of such proteins to make “proteoliposomes, as describedbelow.

A variety of methods are available for preparing a conjugate composed ofa biomolecule and a vesicle-forming lipid. For example, water-soluble,amine-containing biomolecules can be covalently attached to lipids, suchas phosphatidylethanolamine, by reacting the amine-containingbiomolecule with a lipid which has been derivatized to contain anactivated ester of N-hydroxysuccinimide.

As another example, biomolecules, and in particular large biomoleculessuch as proteins, can be coupled to lipids according to reportedmethods. One method involves Schiff-base formation between an aldehydegroup on a lipid, typically a phospholipid, and a primary amino acid onthe biomolecule. The aldehyde group is preferably formed by periodateoxidation of the lipid. The coupling reaction, after removal of theoxidant, is carried out in the presence of a reducing agent, such asdithiotreitol, as described by Heath (1981). Typical aldehyde-lipidprecursors suitable in the method include lactosylceramide,trihexosylceramine, galacto cerebroside, phosphatidylglycerol,phosphatidylinositol and gangliosides.

A second general coupling method is applicable to thiol-containingbiomolecules, and involves formation of a disulfide or thioether bondbetween a lipid and the biomolecule. In the disulfide reaction, a lipidamine, such as phosphatidyl-ethanolamine, is modified to contain apyridylditho derivative which can react with an exposed thiol group inthe biomolecule. Reaction conditions for such a method can be found inMartin (1981). The thioether coupling method, described by Martin(1982), is carried out by forming a sulfhydryl-reactive phospholipid,such as N-(4)P-maleimido-phenyl(butyryl)phosphatidylethanolamine, andreacting the lipid with the thiol-containing biomolecule.

Another method for reacting a biomolecule with a lipid involves reactingthe biomolecule with a lipid which has been derivatized to contain anactivated ester of N-hydroxysuccinimide. The reaction is typicallycarried out in the presence of a mild detergent, such as deoxycholate.Like the reactions described above, this coupling reaction is preferablyperformed prior to incorporating the lipid into the liposome.

Methods for attachment of a biomolecule to the liposome through a shortspacer arm have been described, such as in U.S. Pat. No. 4,762,915. Ingeneral, attachment of a moiety to a spacer arm can be accomplished byderivatizing the vesicle-forming lipid, typically distearolphosphatidylethanolamine (DSPE), with a hydrophilic polymer, such aspolyethylene glycol (PEG), having a reactive terminal group forattachment of an affinity moiety. Methods for attachment of ligands toactivated PEG chains are described in the art (Allen, et al., 1995;Zalipsky, 1992a; Zalipsky, 1992b; Zalipsky, 1993; Zalipsky, 1994). Inthese methods, the inert terminal methoxy group of mPEG is replaced witha reactive functionality suitable for conjugation reactions, such as anamino or hydrazide group. The end functionalized PEG is attached to alipid, typically DSPE. The functionalized PEG-DSPE derivatives areemployed in liposome formation and the desired ligand (i.e.,biomolecule) is attached to the reactive end of the PEG chain before orafter liposome formation.

Another method of linking biomolecules such as proteins to a supportedlipid bilayer is via specific interactions between the side chain of theamino acid histidine and divalent transition metal ions (Malik, et al.,1994; Arnold, 1991) immobilized on the membrane surface. This method hasbeen used, for example, to attach various proteins and peptides to lipidmonolayers (Shnek, et al., 1994; Frey, et al., 1996; Sigal, et al.,1996). Briefly, a cDNA encoding the ligand or receptor which is toimmobilized to the bilayer surface is engineered so that the ligand orreceptor contains a poly-histidine (e.g., hexa-histidine) tag at one ofits termini (e.g., the C-terminus). The bilayer is formed of orderivatized with metal-chelating moieties (e.g., copper-chelatingmoieties or lipids (Shnek, et al., 1994; Frey, et al., 1996)), and theexpressed His-tagged protein is incubated with the vesicles used togenerate the supported bilayer, or with the supported bilayer itself.

Specific high-affinity molecular interactions may also be employed tolink selected biomolecules to a supported bilayer. For example, abilayer expanse may be formed to include biotinylated lipids (availablefrom, e.g., Molecular Probes, Eugene, Oreg.), and a biomolecule linkedor coupled to avidin or steptavidin may be linked to the bilayer via thebiotin moieties.

Biomolecules may also be linked to a supported lipid bilayer viaglycan-phosphatidyl inositol (GPI). The proteins to be linked can begenetically engineered to contain a GPI linkage (Caras, et al., 1987;Whitehorn, et al., 1995). Incorporation of a GPI attachment signal intoa gene will cause the protein to be post-translationally modified by thecell resulting in a GPI linkage at the signal position. It will beappreciated that this type of alteration generally does not affect themolecular recognition properties of proteins such as the ones describedhere (Lin, et al., 1990; McHugh, et al., 1995; Wettstein, et al., 1991).

A convenient approach is to clone the cDNA sequence encoding the proteinof interest into a vector containing the GPI attachment signal usingstandard molecular biology methods and procedures (see, e.g., Ausubel,et al., 1988; Sambrook, et al., 1989). An exemplary vector is thepBJ1Neo derivative described in Whitehorn, et al., (1995), whichcontains a modified polylinker and the human placental alkalinephosphatase (HPAP) GPI linkage signal. Another suitable vector ispBJ1Neo (Lin, et al., 1990). The construct is then transfected intosuitable host cells (e.g., Chinese hamster ovary (CHO) cells) using astandard transfection method, such as electroporation (e.g., usingsettings of ˜0.23 kV/960 μF). Transfected cells are selected, e.g.,using fluorescence activated cell sorting (FACS) with an antibodydirected against the protein of interest.

Transfected CHO cells with high surface expression are expanded inculture. GPI-linked proteins are purified from the cell membranefraction by, e.g., detergent extraction (Schild, et al., 1994). Briefly,almost confluent CHO cells are washed free of medium with PBS containinga cocktail of proteinase and phosphatase inhibitors. The cells are lysedon ice in the same buffer containing 0.5% NP40. Nuclei and cell debrisare spun out and the supernatant is loaded on an antibody affinitycolumn.

The detergent is then exchanged to 1% Octoglucoside (OG) on the column,and the proteins are eluted by base (pH 11.5) containing 1% OG. Afterelution, the proteins are either stored in neutralized elution buffer orthe buffer is exchanged with 1% OG in PBS. The purified GPI-linkedproteins, or any other desired proteins or receptors, may then beincorporated into proteoliposomes as described below.

Proteoliposomes containing a selected membrane protein may be preparedusing standard methods, e.g., using the protocol described by Sadler, etal. (1984). In this method, recombinant receptor proteins areconcentrated in a suitable buffer (e.g., 10 mM Tris pH 8.0, 0.1% LDAObuffer) using, for example, a DEAE ion-exchange column or Centriconconcentrator (Amicon Co., Beverly, Mass.). If desired, the saltconcentration may be adjusted to a desired value (e.g., 100 mM NaCl) bydialysis.

The concentrated receptor proteins are then added to a suspension ofsmall unilamellar vesicles (SUVs; prepared as described below;optionally with a lipid label such as Texas Red), e.g., in a smallconical-bottom vial with stirring, to a selected final RC:lipid moleratio. The ratio is generally between about 1:100 and 1:1000, preferablybetween about 1:300 and 1:500, In one embodiment, the ratio is 1:350.

In the case of the GPI-linked proteins described above, the proteins, atconcentrations of around 100 nM, are mixed with SUVs, at a lipidconcentration of 1 mM, in TN25/50, with the total OG concentrationpreferably not exceeding 0.15%. The detergent may removed by dialysisagainst three changes of 1 liter TN25/50 at 4° C. After dialysis, thelipid concentration may be determined using the NBD-PE absorption at 465nm and adjusted to 0.2 mg/ml.

Alternatively, the samples may be run on a Sepharose column (e.g., aSepharose CL-4B (Sigma) column), previously equilibrated with SUVs tominimize lipid adsorption, and fractions are collected. The absorptionspectra of the proteoliposome fractions are measured, and the trueprotein:lipid mole ratio calculated using the absorption peak of thelipid label.

Typically, the mole ratio of protein:lipid in the fractions follows amonotonic decrease, beginning at about 1:300 and ending at about1:1000-1200. Only the fractions with a mole ratio of about 1:500 orlower are generally used to make planar supported bilayers; thefractions with higher mole ratios do not always form uniform planarbilayers.

IV. Applications

A. Biosensors

In one aspect, the invention includes a biosensor having a surfacedetection array device. The detection array device comprises (i) asubstrate having a surface defining a plurality of distinctbilayer-compatible surface regions separated by one or more bilayerbarrier regions, (ii) a bulk aqueous phase covering the substratesurface, (iii) a lipid bilayer expanse carried on each of thebilayer-compatible surface regions, and (iv) an aqueous film interposedbetween each bilayer-compatible surface region and corresponding lipidbilayer expanse, where each bilayer expanse contains a specie ofreceptor or biomolecule, and different bilayer expanses containdifferent species of receptors or biomolecules. The receptor orbiomolecule is anchored to or in each lipid bilayer expanse. Thespecific binding of a particular ligand to a receptor in a lipid expanseis detected by any of a variety of known biosensor detection mechanisms,such as optical or electrical detection.

In biosensors employing electrical detection, the support gridpreferably contains a conductive electrode and electronic lead for eacharray element of the device. The leads typically terminate as extensionsor “pins” from the device, which can be interfaced with a connectorcable or ribbon leading to a processor. The electrodes preferably format least a portion of the bilayer-compatible surface and are separatedfrom one another by strips of insulating material. They can be used todetect capacitative as well as conductive current transients. In oneembodiment, the electrodes form a portion of the bilayer-compatiblesurface. In another embodiment, the construction of which is detailed inExample 5, the electrodes are positioned just beneath thebilayer-compatible surface, i.e., the electrode surface is coated with athin layer of material, such as low-temperature grown oxide (e.g.,SiO₂), which forms the bilayer-compatible surface. In embodiments wherethis layer is an insulating material, it is preferably less than about 1μm in thickness to enable the detection of capacitative transients casedby binding of ligands to ionophoric receptors. One embodiment of thestructural portion of a surface detection array device suitable for usewith a biosensor is shown in FIG. 4, as described above.

The device is connected to or interfaced with a processor, which storesand/or analyzes the signal from each array element. The processor inturn forwards the data to computer memory (either hard disk or RAM) fromwhere it can be used by a software program to further analyze, printand/or display the results.

Biosensors employing arrays of independently-addressablereceptor-containing lipid bilayer regions have a number of advantagesover previously-available biosensors. For example, the bilayer membranefluidity endows devices of the invention with surface properties similarto those of living cells (e.g., Chan, et al., 1991; Tözeren, et al.,1992). In one particularly compelling set of studies, it was shown thatpurified major histocompatibility complex protein incorporated into asupported membrane can effectively replace the antigen presenting cellin the presentation of a reprocessed antigen to a helper T-cell(McConnell, et al., 1986; Watts and McConnell, 1987).

1. Detection Methods. Receptor-based biosensors operate by detecting thespecific binding of selected analytes to “receptor” biomolecules on thebiosensor. Since the present invention employs fluid bilayers resemblingcell membranes, virtually any transmembrane, membrane-anchored ormembrane-associated protein can be used as the receptor. The receptor isincorporated into the lipid vesicles used to generate the bilayerexpanses of the surface detector array devices. Binding of ligand to areceptor is typically detected either optically orelectrically/electrochemically.

Optical detection methods include ellipsometry (Corsel, et al., 1986;Jönsson, et al., 1985; Vroman and Adams, 1969), optical wave guidance(Nellen and Lukosz, 1990) and surface plasmon resonance (SPR, Cullen, etal., 1988; Liedberg, et al., 1983). SPR is particular advantageous formonitoring molecular interactions in real-time, enabling a sensitive andcomprehensive analysis of the degree of binding interactions between twoproteins.

In this approach, support grid is produced by making a support grid ofan array of conductive regions (e.g., gold) separated by bilayer barrierregions. A very thin polymer film (e.g., polyacrylamide or dextran;Elender, et al., 1996; Khüner, et al., 1994) is then deposited on theconductive regions to form bilayer-compatible surface regions. Khüner,et al., (1994) describe the coupling of polyacrylamide to a surface by3-methacryl-oxypropyl-trimethoxy-silane (MPTS; Serva, Heidelberg,Germany).

Bilayers containing the selected molecules are deposited as described,and the bilayer-containing support grid is placed into a cell whichallows a solution to be passed over the surface containing the array ofreceptor-studded lipid expanses. The grid is illuminated at an anglewith a light-emitting diode (LED), and reflected light is analyzed witha photodetector. Through an evanescent electric field generated by theinteraction of incident light with the gold layer, the reflected lightis sensitive to the environment of a layer extending about 1 μm (□=760nm) from the receptors into the medium. Changes in the environment ofthe receptor, such as are caused by the binding of a ligand to thereceptor, are detected as changes in the reflectance intensity at aspecific angle of reflection (the resonance angle).

Capacitative detection or impedance analysis may also be used. Here, anelectrode is incorporated into each the bilayer-compatible surfaceregion, and a “ground” electrode is placed in the bulk aqueous phase. Avoltage from a variable-frequency function generator is used to generatea selected voltage waveform which is fed across selected array elements.The peak-to-peak amplitude of the voltage is typically on the order ofabout 10 V, but can be substantially less. The voltage is applied over arange of frequencies and the capacitance is determined from the measuredcurrent as a function of signal frequency using standardsignal-processing techniques. Examples of the application of capacitancemeasurements and impedance analyses of supported bilayers are discussed,for example, in Stelzle, et al., (1993) and Stelzle and Sackmann (1989),both incorporated herein by reference.

Other methods of detection are discussed in U.S. Patents relating tobiosensors, including Gitler, et al., 1993; Osman, et al., 1993; Taylor,et al., 1993; Case, et al., 1994; and Tomich, et al., 1994, allincorporated herein by reference.

2. Making of Biosensors. A surface detection array device is producedessentially as described above, except that (i) the vesicles used tomake the bilayer expanse typically contain the desired receptor orbiomolecule (although the receptor or biomolecule may also be introducedafter the bilayer is formed), and (ii) different array elements aretypically made with different vesicle suspensions.

Analyte selectivity is conferred to different array elements bydifferent types of receptors present in the supported bilayer of eacharray element. Such distinct bilayer may be formed using liposomes orproteoliposomes containing the different biomolecules or receptors. Aconvenient method of making such a device is by depositingmicro-droplets of the desired liposome suspension in the differentcompartments of a device substrate housed in a humidified chamber toeliminate fluid loss due to evaporation.

Any of several approaches known in the art can be used to formdifferent-composition bilayers on a single microfabricated support grid.One suitable method employs a modified ink-jet printing device(Blanchard, et al., 1996, incorporated herein by reference) to depositmicro-drops containing selected vesicle suspensions on the individualbilayer-compatible surface regions of the device in a humidifiedchamber. The ink-jet print head of the device is modified to deliversmall drops (e.g., ˜100 μm in diameter) of vesicle-containing suspensionin a high density array format. Adjacent drops may be deposited as closeas 30 μm from one another. The barriers in such applications have awidth that is typically on the order of the minimum separation distancebetween adjacent drops (i.e., ˜30 μm), but can be greater or smaller inparticular applications.

Of course, the vesicle suspensions may also be deposited using standardmicropipeting technology (i.e., a micropipet in a holder connected to amicromanipulator). The micromanipulator may be controlled by a motorizeddrive for greater precision and efficiency. Such drives, as well asmicromanipulators, are commercially available, e.g., from Newport Corp.,(Irvine, Calif.) and Narashige USA, Inc. (Greenvale, N.Y.). The drive inturn can be controlled by a microcomputer for fully automated operation.The entire process can be monitored, if desired, using a conventionalmicroscope, such as a dissecting microscope.

Suitable micropipettes may be made using a standard micropipet puller,such as a puller available from Narashige. The tips of the pipets can bemade to have opening diameters ranging from less than a micron to tensof microns or more. The back of the pipet can be connected to a standardmicroinjection pump set to dispense a desired volume of vesiclesuspension.

The drops containing the vesicle suspensions are allowed to incubate onthe substrate grid for a few minutes to allow essentially all themembranes that are capable of forming to form. The grid is then gentlyflooded with aqueous solution until a suitable bulk aqueous phase isestablished above the bilayer membranes. A convenient method of floodingthe grid without significantly disturbing the bilayers is to bring thelevel of aqueous in the chamber up until the surface is flush with thetop of the grid, but the compartments still contain only theoriginally-deposited drops. The top of the grid is then exposed to afine mist of the aqueous solution until the droplets coalesce into auniform film of aqueous solution. The level of solution is then raisedto a achieve a desired volume of bulk phase aqueous above the grid.

3. Use of Biosensors. A biosensor employing a biosensor surface detectorarray device such as described above can be used to detect lowconcentrations of biologically-active analytes or ligands in a solutioncontaining a complex mixture of ligands. In such a method, the surfacedetector array device is constructed with different receptors in thebilayer expanses at different array positions. To control for signalfluctuations, several different array elements may contain the same typeof receptor. Similarly, designated array elements may be used forpositive and/or negative control purposes.

The biosensor surface detector array device is then contacted with anaqueous solution containing a mixture of ligands to be analyzed for thepresence of selected ligands, such as receptor agonists, where thecontacting takes place via the bulk aqueous solution portion of thedevice. In other words, the mixture to be tested is washed over thedevice, replacing the bulk aqueous portion. When a selected ligandspecifically binds to a receptor, the binding is detected by a suitabledetection method. For example, in an assay for the presence ofacetylcholine (Ach) using an array device containing Ach receptors(AchRs) incorporated into the lipid expanse of at least one arrayelement, the binding of Ach to the AchRs is detected as a change in thetransmembrane voltage or current in the element containing the AchRs.

B. Substrate for Bioactivity Screens

In an embodiment related to the biosensor application described above,devices of the present invention may be used as substrates for holdingan array of receptors employed in bioactivity screens of compounds. Inparticular, high-throughput screens of large libraries of compounds aretypically optimized for speed and efficiency in order to rapidlyidentify candidate compounds for subsequent bioactivity testing. Whensuch bioactivity testing involves, for example, assays for ion channelagonists or antagonist activity, the testing is often done one compoundat a time by a scientist using electrophysiological measurements (e.g.,patch clamping; see, e.g., Hamill, et al., 1981) of individual cellsexpressing the target ion channel or receptor. While this type ofanalysis provides detailed high quality data for each compound, it isslow and inefficient if a large number of compounds are to be assayedfor bioactivity.

Devices of the invention may be used in secondary screen to assess thebioactivity of compounds identified in a high-throughput screen,enabling the scientists to focus on the few truly-interesting compounds.The devices are made essentially as described above for biosensors. Thesame types of binding-detection schemes may be employed, although whenassaying compounds for bioactivity on ionotrophic receptors or ionchannels, electrical detection is typically preferred to opticaldetection.

In devices employing electrical detection using electrodes in each ofthe array elements, it will be appreciated that since a water filmseparates the electrode from the bilayer, an electric field may beapplied across the bilayer membrane, e.g., to activate voltage-dependention channels. This allows screening for compounds which only bind to thechannel when the channel is in a state other than the resting state(e.g., in an activated or inactivated state).

In a related embodiment, devices of the invention are used as substratesfor holding libraries (e.g., combinatorial libraries) of compounds. Thebilayer expanses are deposited onto a support grid from a common bulkvesicle suspension, as detailed in Example 1, and each bilayer expanseregion is then derivatized with a selected biomolecule, e.g., using oneof the methods detailed above. One application if this approach is theuse of light-directed synthesis (Fodor, et al., 1991) to generatespatially-addressable molecular libraries (e.g., peptide libraries) in aform where the peptides are displayed on the surface of the confinedpatches of fluid membrane. This is somewhat analogous to phage display,except that here the peptide sequence is defined by its location in thearray. Such libraries may be particularly useful for cell screening dueto the native-like surface provided by the membrane.

C. Forming Regions with a High Density of Membrane Proteins

The invention also includes a method of forming supported bilayers withregions of very high membrane protein density. As stated above,protein-containing vesicles, or proteoliposomes, can typically only beformed with a protein:lipid mole ratio of about 1:500 or lower—vesicleswith higher mole ratios do not consistently form uniform planarbilayers. Accordingly, high-density arrays of proteins in lipid bilayerscannot be formed by simply fusing protein-containing vesicles with asurface to form a supported bilayer.

As shown in Example 3 and FIG. 3, however, if the supported bilayer isformed in a corral surrounded by bilayer barrier regions and subjectedto an electric field, the membrane proteins can be concentrated intoregions of very high density. This effect can be amplified by, forexample, making the migration focal point the apex of a triangularcorral.

After the proteins have been concentrated, they can be used forsubsequent applications, such as diffraction studies to determinestructure. If desired, the high-density protein region of thefield-induced concentration gradient can be “frozen” by cross-linkingthe proteins using standard cross-linking methods (e.g., treatment withglutaraldehyde).

D. Device for Measuring Receptor Size and/or Aggregation

Another aspect of the invention relates to sorting devices forbiomolecules integrated into or attached to the supported bilayer. Thesorting devices employ the bilayer barrier surface regions not tocompartmentalize the surface into discrete patches, but rather, to actas 2-dimensional sieves having progressively smaller “openings” from oneend of the device to the other. One embodiment of the structural portionof this aspect of the invention is shown in a top view in FIG. 5. Here,the structural portion of the device 70 is formed of a wafer 72 having asubstrate surface 74 defining a bilayer-compatible region bounded on allsides by a bilayer barrier region 76. The bilayer-compatible region isalso interrupted by a plurality of substantially parallel broken lines78 defining bilayer barrier surface regions. The gaps in the lines areof molecular dimensions and get progressively smaller going from oneedge 80 of the device to the opposite edge 82. Electrodes 84, 86 arepositioned near the edges of the device and are connected to a voltagesource 88 via wires 90.

The device is employed to sort membrane-associated molecules by size. Amixture of like-charged molecules having different sizes is loaded inthe well formed by the bilayer barrier region circumscribing and thebroken line having the largest gaps. The voltage source is turned onwith a polarity to cause the charged biomolecules to migrate through theprogressively smaller gaps of the consecutive barriers until they gettrapped according to size in the well defined on the “downstream” sideby a barrier having gaps too small for the molecules to pass through.

In a related application, the bilayer barrier regions are arranged toprovide a uniform or graded array or network of barriers, andelectrophoresed membrane molecules are sorted based on the migrationtime through the array. Here, the method of separation is similar tothat obtained with a gel, such as an agarose or polyacrylamide gel,where smaller molecules migrate faster than larger molecules.

In another aspect of the invention, the biosensor array furthercomprises a plurality of biosensors where one or more biosensors in thearray has a bilayer composition different than the remaining otherbiosensors present within the biosensor array. The resulting gradientbiosensor array forms at least a one-dimensional or two-dimensionalgradient across it with respect to the local concentration of bilayercomposition upon each biosensor. Although at the initial formation ofthe gradient, a sub-gradient may exist across any one particularbiosensor, such sub-gradient rapidly homogenizes within the confines ofthe lipid bilayer region captured by a particular bilayer-compatibleregion situated under each bilayer.

As discussed above, when lipid-bilayer-forming compositions containinglipid vesicles are contacted with a bilayer-compatible region that issurrounded by one or more bilayer-barrier regions, the vesicles fuse ateach bilayer-compatible region surface to form a continuous bilayerexpanse thereabove. Because each bilayer expanse is separated from theother by the presence of the bilayer-barrier regions, the resultingbilayer expanses supported above each region are in two-dimensionalmatrix isolation from one another. Consequently, the compositions ofdifferent, yet adjacent bilayer expanses above bilayer-compatibleregions will remain distinct despite their close proximity. Such resultprovides the unexpected advantage of allowing a user to rapidly andreliably create a biosensor array having a plurality of differentbiosensor regions, each region having a distinctly different bilayercomposition from its adjacent bilayer expanse. Bilayer compositionmeans, for the purpose of this section, both the chemical components ofthe bilayer itself, and any other components that are lipid-deliverableduring delivery as lipid-bilayer forming vesicles, for example, receptorsubunits, different receptors, other cell membrane communicationfactors, and the like. Differing ratios of receptor subunits may be“titered” out using this method. Additionally, the method can be used tocreate different ratios of bilayer forming components, for exampledifferent phospholipids.

A gradient biosensor array may be created by contacting differentbilayer compositions with different bilayer-compatible regions within abiosensor array such that each different composition remains within itsrespective bilayer-compatible region and is separated from other,different bilayer composition containing bilayer-compatible regions byone or more bilayer-incompatible regions. One method includesselectively contacting different bilayer-forming compositions withdifferent areas of a biosensor array that each contain a plurality ofbilayer-compatible regions separated from one another by one or morebilayer-barrier regions resulting in a biosensor array with a pluralityof different areas each containing a plurality of bilayer-compatibleregions having the same composition within such area, but different fromother areas within the biosensor array.

Gradients can be formed in a variety of ways. For example, a simpletwo-dimensional gradient can be formed by drawing a mixture from atleast two containers in fluid communication with one another through asmall diameter bore channel such as a capillary tube. In its simplestform, two bottles placed side by side, may be connected by a small boresiphon tube, and fluid removed from the one of the two bottles of thetwo bottle system by yet another tube via pumping or siphonic action.Each bottle contains a different concentration of the component thatwill form the gradient. Depending on which bottle is drawn from, eitherthe high concentration or low concentration, a high to low, or a low tohigh effluent gradient is formed as the two-bottle system is drawn upon.The effluent may then be further passed through a wide spreading-thinprofile nozzle to coat a surface of a biosensor array. By moving thearray laterally with respect to the nozzle, a gradient of mixturesacross the biosensor surface is formed. Gradient forming devices areknown in the art, for example, as found in U.S. Pat. Nos. 3,840,040,4,074,6878, and 4,966,792, each included entirely by reference herein,which may be further adapted in accordance with the present invention toyield a device for forming a gradient biosensor array.

Yet another aspect of the invention provides for a method for forming anarray of biosensor regions, where each region has a different, knownlipid bilayer composition comprising the steps of providing a biosensorarray having a plurality of lipid bilayer compatible regions, eachcompatible region being surrounded by one or more bilayer barrierregions, providing a gradient forming devices loaded with two or moredifferent lipid bilayer compositions, the gradient forming device influid communication with a spot forming device for forming spots on asurface, providing a multi-axis translation table for holding andtranslating a biosensor array workpiece, and placing a biosensor arrayworkpiece that has a plurality of bilayer compatible regions surroundedby one or more barrier regions, and forming spots of mixed lipid bilayercompositions resulting from the gradient forming device forming agradient and translating the table in at least one axis while dispensingsuch composition mixture as it is formed thereby dispensing todifferent, consecutive locations different ratios of eachn of the lipidbilayer compositions. Thus, a gradient of lipid bilayer compositions isformed with respect to the ratio of each composition, the result of suchgradient is distributed across the array of biosensors by rasterscanning the relative position of the spot forming device's outputacross the surface of the array and thereby depositing at differentbiosensor locations, different composition mixtures.

In yet another embodiment of the invention, a gradient may be formedacross a surface of an array of biosensors in one dimension. A methodfor making a gradient biosensor array comprises the steps of

mixing together first and second different lipid bilayer formingcompositions contained from first and second sources by flowing in asubstantially laminar flow, two different compositions from twodifferent sources into one mixing chamber that substantially retains thelaminar flow character of the two different compositions while flowingthrough the mixing chamber, where the facing edges of each differentcomposition mix to form a gradient having a first edge and a second edgeand further comprising composition combinations of different ratiosbeginning from the first edge of the gradient that faces the firstcomposition, and ending at the second edge of the gradient that facesthe other, second composition, and where the mixing chamber is adaptedto dispense the gradient in a substantially laminar flow across thesurface of the array, and

where the compositions contained in the gradient are captured andretained upon initial contact by bilayer-compatible regions of thearray. Other embodiments may further comprise the mixing chamber beingthe surface of the biosensor array where the first and secondcompositions are supplied to the surface by a plurality of sourcesadjacent to the array, each containing a different composition. Further,other embodiments may further comprise a plurality of differentcompositions contained within a plurality of different sources in fluidcommunication with the mixing chamber.

In accordance with the present invention, lipid bilayers spontaneouslyform over lipid bilayer regions distributed across the array. Forexample, as a gradient mixture is dispensed from a gradient formingdevice, and such mixture contacts a biosensor array containing aplurality of bilayer-compatible regions surrounded by one or morebilayer-barrier regions, components of the mixture, such as lipidbilayer vesicles optionally containing other bilayer components, contactthe bilayer-compatible regions and spontaneously form continuous lipidbilayers adjacent to each bilayer-compatible region, but with each suchcontinuous bilayer being discontinuous from other continuous bilayerregions, separated from one another by bilayer-incompatible regions. Theattractive, bilayer forming forces associated with each bilayercompatible region serves to capture from the gradient mixture theinstant mixture initially present to the bilayer-compatible region thusforming and retaining the mixture character of the gradient mixtureinitially presented to such bilayer-compatible region despite subsequentpresentation of gradient mixtures different than what was initiallypresented to the bilayer-compatible region during the formation of thegradient biosensor array. Once a lipid-bilayer region is formed, itscomposition, in general, is not susceptible to change if exposed tolipid bilayer forming compositions.

In still yet another embodiment of the invention, a plurality ofgradient forming devices are in fluid communication with one or moresecondary gradient forming devices where a first gradient is combinedwith one or more different gradients to form a complex gradient formingdevice in fluid communication with a distribution devices fordistributing a gradient mixture across an array of biosensors.

FIG. 6 depicts a top down view of the forming of a gradient biosensorarray. Lipid bilayer forming composition A combines with lipid bilayerforming composition B at point 603 in mixing device 605 to form asubstantially laminar flow gradient flowing in direction 607. At acertain point, indicated by line 609, the cross sectional profile of themixture flow or effluent may be characterized by graph 611 showing thecross sectional concentration of both A and B at each point across thecross section. Graph 613 represents the bilayer composition at eachdistinct bilayer-compatible region within the gradient biosensor array.

FIGS. 7a-d depict, in color, the formation of a two-composition gradientbiosensor array. In FIG. 7a, biosensor array 701 is slightly biasedupward on one end with respect to gravity. In FIG. 7b, first bilayerforming component 703, TEXAS RED, is flowed onto array 701's surface 701a, while second bilayer forming component 705, NBD in green, is flowedonto surface 701 a. The resulting flow streams from each component forma laminar flow across array 701 as indicated by arrows 709 a and 709 b.The resulting grid in FIG. 7d is graphically portrayed in FIG. 7c fromdata collected from spectral intensity analysis of the array in onedimension.

The following examples illustrate but in no way are intended to limitthe present invention.

Materials and Methods

Unless otherwise indicated, chemicals were purchased from Sigma (St.Louis, Mo.) or United States Biochemical (Cleveland, Ohio).

A. Buffers

Standard Buffer

10 mM Tris

100 mM NaCl (pH 8.0)

Phosphate-buffered Saline (PBS)

10×stock solution, 1 liter:

80 g NaCl

2 g KCl

11.5 g Na₂HPO4—7H₂O

2 g KH₂PO₄

Working Solution of PBS, pH 7.3:

137 mM NaCl

2.7 mM KCl

4.3 mM Na₂HPO₄—7H₂O

1.4 mM KH₂PO₄

B. Lipids and Labels

L-α phosphatidylcholine from egg (egg-PC) were obtained from AvantiPolar Lipids (Alabaster, Ala.). The fluorescent probe N-(Texas Redsulfonyl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine,triethylammonium salt (Texas Red DHPE) was obtained from MolecularProbes (Eugene, Oreg.).

C. Preparation of Phospholipid Vesicles

Small unilamellar vesicles (SUVs) were prepared by following theprotocol outlined in Barenholz, et al. (1977) using egg L-αphosphatidylcholine (Avanti). The phosphatidylcholine was mixed with 1mole % Texas red DHPE in HPLC-grade chloroform (Sigma-Aldrich) and driedin a vacuum desiccator overnight. The dried lipids were resuspended toabout 6 mg/ml in standard buffer which had been filtered through RaininNylon-66 0.45 □μm filters using a Sibata filter unit. The suspension wassonicated to clarity with a Branson ultrasonicator under flowing Ar onice for 3 minute periods separated by 1 minute cooling periods (Martin,1990). The sample was then spun for 30 minutes at 100,000×g to remove Tiparticles from the sonicator tip, and the supernatant was spun for 4hours at 166,000×g to obtain the SUVs. The SUVs were stored at 4° C.under N₂ or Ar in the dark and were used within three weeks. The lipidconcentration in these samples was determined from the Texas Red probeabsorption at 590 nm (ε=100,000 M⁻¹cm⁻¹; Haugland, 1992) assuming thatthe probe concentration in the vesicles is 1 mole % as prepared. Yields(mg SUV lipid/mg initial lipid) are calculated from this concentrationand are equal to those reported by Barenholz, et al. (1977).

D. Membrane Electrophoresis

For the electrophoretic studies, the supported membrane in PBS wasdiluted to 1 mM total ionic strength. This was then assembled, underbuffer, into a sandwich with another coverslip. The electrophoresis cellconsisted of two 0.01″ diameter platinum wire electrodes insolution-filled wells of a Teflon trough. The coverslip sandwich wasarranged to form a bridge between the two electrode wells. Electricalconnection was achieved through the solution in the cover slip sandwich.Fields up to 60 V/cm were applied with a standard power supply. Currentswere monitored with a Keithley picoammeter (Cleveland, Ohio) and weretypically around 3 μA for a single 18 mm square coverslip sandwich at 15V/cm. This corresponds to a total power dissipation of 9×10⁻⁵ W whichshould produce a negligible amount of Joule heating.

EXAMPLE 1 Construction of a Surface Detector Device

A patterned support grid was microfabricated using standard techniques(Wolf and Tauber, 1986). 100 mm diameter silicon 1-0-0 wafers wereobtained from Silrec Corporation (San Jose, Calif.). The wafers weremaintained in steam at 1000° C. in an oxidation furnace (Tylan Inc., SanDiego, Calif.) to generate a ˜1 μm thick layer of thermal oxide.Standard positive photoresist (S-1800; Shipley Inc., Marlborough, Mass.)was spun onto the wafers at a thickness of one micron with a trackcoater (Silicon Valley Group, San Jose, Calif.).

The wafers were exposed for ˜8 seconds to ˜10 mW/cm² UV light through aphotolithographic mask with a contact mask aligner (Karl Suss America(Waterbury Center, Vt.), MA-4). Development was done on a trackdeveloper (SVG) using standard tetramethylammonium hydroxide(TMAH)-based developer (Shipley). The wafers were then subjected to athree minute etch in argon plasma.

Membranes were formed by contacting the patterned surface of the wafersupport grids with a suspension, prepared as described above, containing˜25 nm diameter unilamellar vesicles consisting primarily ofL-α-phosphatidylcholine (PC) molecules doped with 1 mole percent of thefluorescently labeled lipid, Texas Red DHPE. Vesicles in the suspensionspontaneously assembled in a matter of seconds to form a continuoussingle bilayer on the bilayer-compatible regions of the support grid, asevidenced by photobleaching and electrophoresis experiments describedbelow.

Excess vesicles were rinsed away while maintaining the membrane underthe bulk aqueous solution at all times. Results of extended experimentsmonitoring the state of the supported bilayers indicated that thebilayers are stable under water and retain their uniformity and fluidityfor a period of weeks.

EXAMPLE 2 Fluidity of Supported Bilavers Assayed by Photobleaching

Long-range fluidity within the bilayer-compatible regions, or corrals,was observed by fluorescence recovery after photobleaching (FRAP). Theexperiment is described with respect to FIGS. 2A and 2B, which showschematics of a surface detector array device containing 25bilayer-compatible surface regions, or corrals, with a correspondinglipid bilayer expanse carried on each of these surface regions. Thedevice was made from an oxidized silicon wafer patterned withphotoresist to generate corrals dimensioned 100 μm per side. The 10 μmwide photoresist (bilayer barrier regions) appears as the blackboundaries separating the 25 corrals in FIGS. 2A and 2B. Texas Red DHPElipid probe (Molecular Probes, Eugene, Oreg.) was incorporated in thebilayer membrane to serve as a label.

A circular beam of light having a diameter of less than 100 Am was usedto photobleach the fluorescent probe molecules in five individualcorrals 40 (FIG. 2A), yielding the results schematized in FIG. 2B.Diffusive mixing of molecules within each corral caused the circularbleached spot to spread, filling the square corral. The lines ofphotoresist acted as barriers to lateral diffusion, preventing mixingbetween separate corrals. Fluidity of the membrane was evidenced by thespreading of the bleached region to fill each square corral. If themembrane had not been fluid, a circular bleached (dark) region wouldhave remained.

Bleach patterns such as those illustrated in FIG. 2B were stable formany days, whereas spots photobleached into a single continuous membranewith no such barriers diffused away completely in about 30 minutes.

EXAMPLE 3 Fluidity of Supported Bilayers Assayed by Electrophoresis

The fluidity of the supported bilayers on the bilayer-compatible surfaceregions was also assessed by electrophoretic redistribution of chargedmembrane components. This method illustrates both the fluidity of thelipid bilayer and confinement of different-composition bilayer patchesto distinct independently-addressable bilayer-compatible surfaceregions.

A device with 200 μm square corrals was prepared as described aboveusing PC molecules doped with 1 mole percent of the fluorescentlylabeled lipid, Texas Red DHPE (Molecular Probes).

An electric field of 15 V/cm was applied parallel to the lipid bilayermembrane. Upon application of the field, the charged molecules (labelledDHPE) drifted in the plane of the bilayer, whereas the neutral PCmolecules, forming the bulk of the membrane, were unaffected by thefield. Application of the field for ˜25 minutes resulted in asteady-state, electric field-induced concentration profile (Groves andBoxer, 1995) of the negatively-charged fluorescent probe.

A quantitative description of the field-induced concentration gradientis depicted in FIG. 3, which shows quantitative traces of fluorescenceintensity calculated from videomicrographs of steady-state concentrationgradients of the fluorescent probe lipid (Texas Red DHPE) in two 200 μmmicrofabricated corrals. The concentration gradients in this experimentadopted an exponential profile. The image from which the fluorescenceintensity traces were calculated was taken with a low light level videocamera which had been adjusted for linear imaging of fluorescenceintensity.

The field-induced concentration gradients were fully reversible, takingapproximately the same amount of time to dissipate as they took to format 15 V/cm. The profiles could be switched by reversing the polarity ofthe field repeatedly without any apparent effect on the membrane or thebilayer-barrier regions, or barriers. The field-induced concentrationprofiles described above can be used to study molecular size,clustering, and non-ideal mixing.

EXAMPLE 4 Bilayer Barrier Regions Do Not Function by MechanicallySeparating Adjacent Bilayer Expanses

Experiments were performed to determine whether the bilayer barrierregions isolate adjacent bilayer expanses by mechanical separation or byintrinsic properties of the material making up the bilayer barriersurface regions. Bilayer membranes were deposited on unpatterned SiO₂substrates (i.e., a substrate having a single bilayer-compatiblesurface) as described above. However, the topography of thebilayer-compatible surface was the same as that of thephotoresist-patterned SiO₂ substrate described above.

Continuity of the bilayer(s) was assayed using the FRAP andelectrophoretic methods described above. The results indicated that thelipid expanse was a single supported membrane which followed thecontours of the corrugated surface without disruption.

EXAMPLE 5 Generation of an Array Device with Electrodes Under theSupported Bilavers

This example describes the making of silicon electrodes covered by athin layer of silicon dioxide. Bond pads may be used to connect wires tothe silicon electrodes.

A. Wafers

Silicon-on-oxide wafers were purchased from Ibis Technology Corporation,Danvers, Mass. The wafers are 100 mm in diameter and approximately 500μm thick. As supplied by the manufacturer, the wafers have a ˜0.4 μmthick silicon dioxide layer buried under a ˜0.2 μm thick layer of puresilicon, which forms the top surface of the wafer.

B. Pre-resist Clean and Resist Coating

The wafers were cleaned with a conventional RCA cleaning procedure (Kernand Puotinen, 1970; Wolf and Tauber, 1986, p. 516), baked at 150° C. for30 minutes, and coated with 1 μm of photoresist (Shipley S-1813) usingconventional spin coating with a Silicon Valley Group (SVG) track coatersystem.

C. Exposure and Development

The mask pattern was exposed using an 8 second exposure on a Karl SussMA-4 contact mask aligner with an electron beam master mask consistingof chrome patterns on a quartz substrate. The wafers were then soaked inchlorobenzene for 15 minutes before developing with standard TMAH(tetramethylammonium hydroxide) based developer (Shipley) using aSilicon Valley Group track developer system.

D. Etch and Thin Oxide Growth

The electrode patterns were etched into the top silicon layer using aconventional fluorine based plasma etch (Wolf and Tauber, 1986), whichselectively etches silicon, but not silicon dioxide. The gases used inthe plasma etch were SF₆, O₂, and CHF₃. Following a second RCA clean, athin oxide was grown at 1000° C. in a steam oven to a thickness of 0.1μm.

E. Pattern for Bond Pads

Another RCA clean is performed and a new layer of resist is deposited asdescribed above. A new pattern, which defined openings in the oxidelayer grown in the previous step, was transferred to the resist byphotolithography as described above and the exposed resist was developedas described above.

F. Etch Openings for the Bond Pads

The wafers were etched in an Applied Materials (Santa Clara, Calif.)reactive ion etcher to open holes in the top oxide layer so thatcontacts to the underlying silicon layer could be made for bond pads.

G. Evaporate Gold

A 0.3 μm layer of gold was evaporated on the wafer before the resistfrom the previous step was removed. This gold was then lifted off withacetone, resulting in gold bond pads located in the holes which wereetched in the previous step.

While the invention has been described with reference to specificmethods and embodiments, it will be appreciated that variousmodifications may be made without departing from the invention.

It is claimed:
 1. A method for forming an array of biosensor regions,where each region has a different, known lipid bilayer compositionscomprising the steps of: providing a biosensor array having a pluralityof lipid bilayer compatible regions, each compatible region beingsurrounded by one or more bilayer barrier regions, providing a gradientforming device loaded with two or more different lipid bilayercompositions, the gradient forming device in fluid communication with aspot forming device for forming spots on a surface, providing amulti-axis translation table for holding and translating a biosensorarray workpiece, placing a biosensor array workpiece that has aplurality of bilayer compatible regions surrounded by one or morebarrier regions, and forming spots of mixed lipid bilayer compositionsresulting from the gradient forming device forming a gradient andtranslating the table in at least one axis while dispensing suchcomposition mixture as it is formed, thereby dispensing to different,consecutive locations different ratios of each of the lipid bilayercompositions.
 2. A method for making gradient biosensor array comprisingthe steps of: mixing together first and second different lipid bilayerforming compositions contained from first and second sources by flowingin a substantially laminar flow, two different compositions from twodifferent sources into one mixing chamber that substantially retains thelaminar flow character of the two different compositions while flowingthrough the mixing chamber, where the facing edges of each differentcomposition mix to form a gradient having a first edge and a second edgeand further comprising composition combinations of different ratiosbeginning from the first edge of the gradient that faces the firstcomposition, and ending at the second edge of the gradient that facesthe other, second composition, and where the mixing chamber is adaptedto dispense the gradient in a substantially laminar flow across thesurface of the array, and where the compositions contained in thegradient are captured and retained upon initial contact bybilayer-compatible regions of the array.